Plague and its laboratory diagnosis

Plague is an acute bacterial infection in man caused by Yersinia pestis. Historically, it has resulted in devastating pandemics (Black Death) throughout the world with high mortality rates in which pneumonic man-to-man transmission followed the usual flea-to-man transmission [1]. Plague may have retreated over the past decades due to availability of effective antibiotics and intensive vector control, but it has not been completely eradicated [2], and the third pandemic can be regarded as still ongoing, since Y. pestis sporadically re-emerges from its reservoir of wild rodents and spreads to the human population [3]. During the last 15 years more than 20 countries have reported plague cases to the WHO [3].

Outbreaks of plague in India emphasise the need to maintain the necessary skills and public health infrastructure to detect, monitor and combat a wide range of in infectious disease agents. Y. pestis is spread by aerosol and is usually fatal if treatment is not started within 24 hours of onset of symptoms [3,4]. A precise and quick diagnosis of plague allows prompt intervention, especially necessary during plague outbreaks. When rapidly diagnosed and promptly treated, plague may be successfully managed with antibiotics, reducing the mortality from 60% to less than 15% [5,6].

Clinical features
The diagnosis of plague still relies on clinical symptoms and exposure history in most endemic areas [1,3]. The three main clinical presentations of plague are bubonic, septicaemic and pneumonic plague. Bubonic plague is the classical form of the disease, characterised by fever, headache, chills and swollen, extremely tender lymph nodes or buboes within 2-6 days after contact with the pathogen. Buboes typically involve lymph nodes that drain the site of initial infection and are usually located in the inguinal, axillary or cervical region. In septicaemic plague, patients have positive blood cultures without palpable lymphadenopathy. Clinical symptoms, such as chills, headache, malaise and gastrointestinal disturbances resemble septicaemia caused by other gram negative bacteria. Pneumonic plague is the rarest form of plague with the highest mortality rate. Signs of pneumonic plague include severe pneumonia accompanied by high fever, dyspnea and often haemoptysis.

Laboratory diagnosis
Laboratory diagnosis of plague is mainly based on bacteriological and/or serological evidence. However molecular biological techniques based on PCR and DNA hybridisation have also been developed in recent years. Plague should be suspected if: clinical symptoms are compatible with plague i. e., fever and lymphadenopathy in a person who resides in or has recently travelled to a plague-endemic area; and small gram-negative and/or bipolar-staining coccobacilli are seen on a smear taken from affected tissues, e.g. bubo (bubonic plague), blood (septicaemic plague) and tracheal/lung aspirate (pneumonic plague).

The presumptive diagnosis of plague can be made if: an immunofluorescence stain of a relevant sample is positive for the presence of Y. pestis Fraction 1 capsular (F1) antigen; and a single serum specimen is tested and the anti-F1 antigen titre by agglutination is >1:10.* Confirmed plague is diagnosed if: an isolated culture is lysed by specific bacteriophage; two serum specimens demonstrate a four fold anti-F1 antigen titre rise by agglutination testing;* and a single serum specimen tested by agglutination has a titre of >1:128 and the patient has no known previous plague exposure or vaccination history.*
*Agglutination testing must be shown to be specific to Y. pestis F1 antigen by
haemagglutination inhibition.

Bacteriological work-up
This includes microscopy, isolation by cultivation, identification and confirmation by NAT tests and animal pathogenicity tests for Y. pestis. Specimens should be obtained from relevant sites at the appropriate time to enhance the reliability of the results. The preferred specimen for microscopic examination and isolation from a bubonic case is pus from an accessible bubo, which contains numerous organisms. Blood cultures should be taken whenever possible, particularly in septicaemic plague. Bronchial/tracheal washing should be taken from patients suspected of pneumonic plague. Throat swabs are not ideal for isolation of plague bacilli since they often contain many other bacteria that can mask the
presence of plague organisms [9].

Staining techniques such as Gram, Giemsa, Wright or Wayson stains can provide supportive but not confirmatory evidence of plague [2]. Blood smears taken from suspected bubonic plague patients are frequently negative as the bacteria are intermittently released from lymph nodes; periodic specimen collection at 10-30 minutes intervals gives more reliable results [9]. Sputum or throat smears taken from pneumonic plague patients may contain too many other organisms which can mask plague if only the staining techniques mentioned above are used. In addition smears should be stained using the more specific fluorescent antibody test (FA), which utilises fluorescent dye and targets capsular F1 antigen expressed predominantly at 37° C [2,9]. A positive fluorescent antibody test can be used as presumptive evidence of Y. pestis infection however it necessitates a specialised instrument along with costly reagents. Samples that have been refrigerated for more than 30 hours or collected from cultures that were incubated at temperatures lower than 35°C (from fleas, for instance) may be negative [2].

Specimens intended for culture should be taken before antibiotic therapy is started [2]. Y. pestis takes two days to form visible, hammered metal-like colonies. If patients have been treated with a bacteriostatic antibiotic for more than four days, bacterial cultures should be incubated for more than five days to allow the remaining organisms to build up a population. A single colony from the culture is then tested for Y. pestis using biochemical tests, inoculation into laboratory animals and use of specific bacteriophage. All automated microbiological test systems are not programmed to identify Y. pestis and its low growth rate restricts the use of biochemical identification [2]. Lysis by specific bacteriophage is used by the CDC to conclusively identify Y. pestis [2], but all these procedures are too complex and time-consuming, and also expensive. Furthermore, incorrect handling while shipping the specimens from the collection centre to the diagnostic centres may lead to desiccation, contamination of the samples and consequent death of the bacteria [6].

Serological analysis
Serological tests are useful especially when cultures yield negative results [9] and/or only serum samples are available as clinical specimens. Such tests are often used retrospectively to confirm the diagnosis of plague; paired serum samples are collected during either the acute and convalescent phases or the convalescent and post-convalescent phases. Serological tests are primarily based on Passive Haemagglutination (PHA) tests and ELISA [10]. PHA has been routinely performed for the last 30 years for the serodiagnosis of plague according to the procedure described by the WHO (1970), which advocates the use of formaldehyde-fixed and F1 sensitised sheep RBC. Being a simple, cheap, rapid and sensitive method, it is regularly practiced at CDC to analyse samples for the presence of anti-F1 antibodies. A four-fold rise in the titre is considered confirmatory for plague. A single serum sample with a titre greater than 1:10 in a person not previously infected or vaccinated against plague is presumptive evidence of recent infection [2]. However, PHA has proved to be an invaluable tool in surveillance programmes for large scale screening [11].

Using monoclonal antibody as a capture antibody and purified rabbit immunoglobulin as an indicator, Williams et al described an ELISA that detects up to 8 ng of F1 antigen per mL serum [12]. ELISA has been used to measure the level of F1 antigen as well as IgG and IgM antibodies to F1 antigen. Detection of IgM antibodies by ELISA in convalescent phase sera is indicative of recent infection. However because of the low concentration of IgM antibody relative to IgG, it is conceivable that some early convalescent phase sera may be IgG positive but IgM negative [12]. Even though it is efficient for serodiagnosis, plague ELISA is not widely accepted because it is less sensitive than FA [12], difficult to perform in the field, and requires specific equipment and expensive reagents.

As well as the two tests described above, tests such as complement fixation and immunodiffusion tests have also been used for plague diagnosis, but have not been widely accepted due to their long turn around time and greater complexity [12]. Furthermore the rarely encountered cases with weak F1 antigen expression also limit the sensitivity of serodiagnostic tests for plague. The latest developments in immunological test design have resulted in availability of rapid diagnostic tests (RDT for plague diagnosis, discussed later in this article.

Nucleic acid tests
When live organisms are not available, e.g., in specimens taken postmortem from lymphoid tissues, lung and bone marrow, DNA of Y. pestis can still be detected [2, 9, 10]. Such methods include PCR and DNA hybridisation techniques. PCR, utilising primers derived from the pla and caf 1 genes that are contained in two different Y. pestis virulence plasmids, can detect levels as low as 10-50 bacteria and thereby help in presumptive diagnosis of plague [10]. However the sensitivity of the method was found to be low when it was used in a field study [13]. This may have been due to suboptimal field conditions and the small volumes of samples used for DNA extraction [13]. Several methods for amplification of Y. pestis DNA have been developed, but none of them has been evaluated with clinical specimens. In spite of its probable usefulness in field studies, DNA hybridisation techniques are of minimal value due to their low limit of detection (minimum of 105 bacteria) and the longer time required to perform the tests [2].

Rapid tests
Even though a number of presumptive and confirmative techniques are available, none are sufficiently simple, economical and non-instrumental to be used routinely by clinical laboratories and in field studies, surveillance or point-of-care testing [2]. The RDT for plague based on F1 antigen has been tested in laboratories and has provided promising results. It is as specific as and at least as sensitive as the available standard methods for plague diagnosis. The excellent specificity of RDT coupled with its lower detection threshold makes it a very useful screening test, in addition to bacteriological tests and ELISA [4].

Span’s Crystal Ypes is a simple and cost-effective RDT based on the principle of immunochromatography. As in the company’s other immunochromatography tests, monoclonal antibodies labelled with a visually detectable marker viz. colloid gold bind to the target antigen. The complex is arrested at the test area (band) coated with another monoclonal antibody and forms a visible band. To the best of our knowledge, it is the only commercially available RDT to diagnose plague at present. The test detects the F1 antigen specific for Y. pestis, which is present in large amount in sputum and bubo samples of infected people. The F1 antigen is stable in tropical climates and can be still be detected in patients after several days of treatment [4]. F1 antigen is detected at a wider range of concentration than other tests, within 10-15 minutes, without the prozone phenomenon, thus avoiding false negative results especially in post mortem specimens or sputum specimens which can contain a very high concentration of F1 antigen [4]. The striking feature of Crystal Ypes is the innovative design of its sample processing device, which minimises the laboratory operator’s contact with highly contagious clinical specimens such as bubo aspirates and tracheal wash without compromising the ease and simplicity of carrying out the test. Because the F1 antigen is highly stable, and there is a reasonably good detection threshold, a short time to result and the test is cost-effective, it is the most suitable test for the immunodetection of plague even in anthropological applications such as burial site studies, especially when archeological and historical data are incomplete or non-existent [14,15].

1. Butler T et al. Journal of infectious disease 1977; 136(2): 317-320.
2. Perry RD and. Fetherston JD. Clinical Microbiology reviews 1997; 10(1): 35-66.
3. Thullier P et al. Am J Med Hyg 2003; 69(4): 450-451.
4. Chanteau S et al. The lancet 2003; 361: 211-216.
5. WHO programmes and projects global alert and response, disease covered by EPR, Plague, 2009.
6. Leal NC, de Almedia MP. Rev Med trop S Paulo 1999; 41(6):339-342.
7. Infectious disease epidemiology section office of Public health, Louisiana Dept of Health and hospitals, plague, 2004.
8. CDC report, Division of vector born disease, Plague, Laboratory test criteria for diagnosis of plague, 2005.
9. CDC, Plague diagnosis CDC Division of vector born infectious diseases, 2005.
10. Murry PR et al. Manual of Clinical Microbiology, 8th Ed. ASM press, 2003, 673-683.
11. de Almeida AMP and de Souza FLC. Mem Inst Inst Oswaldo Cru [online]. 1992; 87 n.1 ISSN 0074-0276. [Online]. 1992; 87 no.1 ISSN 0074-0276
12. Shepherd AJ et al. Journal of Clinical Microbiology 1986; 24(6):1075-1078.
13. Rahalison L et al. Institut Pasteur de Madagascar, Antananarivo, Madagascar 1999.
14. Bianucci R et al. American Journal of Physical Anthropology 2008; 136(3):361-7
15. Bianucci R et al. C R biologies 2007; 33: 0747-754.

Span Diagnostics Ltd
Udhna, Surat, India